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The Hepatitis B Virus X Protein Disrupts
Innate Immunity by Downregulating
Mitochondrial Antiviral Signaling Protein
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of May 9, 2017.
Congwen Wei, Caifei Ni, Ting Song, Yu Liu, XiaoLi Yang,
Zirui Zheng, Yongxia Jia, Yuan Yuan, Kai Guan, Yang Xu,
Xiaozhong Cheng, Yanhong Zhang, Xiao Yang, Youliang
Wang, Chaoyang Wen, Qing Wu, Wei Shi and Hui Zhong
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Copyright © 2010 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 185:1158-1168; Prepublished online 16
June 2010;
doi: 10.4049/jimmunol.0903874
http://www.jimmunol.org/content/185/2/1158
The Journal of Immunology
The Hepatitis B Virus X Protein Disrupts Innate Immunity by
Downregulating Mitochondrial Antiviral Signaling Protein
Congwen Wei,*,1 Caifei Ni,*,1 Ting Song,*,1 Yu Liu,† XiaoLi Yang,† Zirui Zheng,*
Yongxia Jia,* Yuan Yuan,* Kai Guan,* Yang Xu,‡ Xiaozhong Cheng,* Yanhong Zhang,*
Xiao Yang,* Youliang Wang,* Chaoyang Wen,x Qing Wu,x Wei Shi,‡ and Hui Zhong*
H
epatitis B virus (HBV), a member of the Hepadnaviridae
family, is a hepatotropic noncytopathic DNA virus that is
estimated to infect 300 million people, with a particularly
high prevalence found in Asia and Africa. Approximately 5% of the
infected individuals develop chronic infection. Most chronically infected patients remain largely asymptomatic without life-threatening
liver disease, but 10–30% of the patients develop liver cirrhosis with
possible progression to liver cancer. Mechanisms involved in viral
clearance and persistence remain elusive (1).
The innate immune system, which is characterized by production
of type I IFN-a/b cytokines and activation of NK cells, plays
important roles in the detection and elimination of invading
pathogens. The host senses viral and bacterial pathogen invasion
by recognition of pathogen-associated molecular patterns with
pattern recognition receptors, including membrane-bound TLRs
*Beijing Institute of Biotechnology and †The General Hospital of Chinese People’s
Armed Police Forces; xChinese People’s Liberation Army General Hospital, Beijing;
and ‡Key Laboratory for Molecular Enzymology and Engineering, JiLin University,
Changchun, China
1
C.W., C.N., and T.S. contributed equally to this work.
Received for publication December 8, 2009. Accepted for publication May 16, 2010.
This work was supported in part by National Natural Science Foundation of China
Grants 30772605, 30700413, 30870500, and 30871276 and in part by Beijing Natural
Science Foundation Grant 7092081.
Address correspondence and reprint requests to Drs. Hui Zhong and Wei Shi, Beijing
Institute of Biotechnology, Beijing, China. E-mail addresses: [email protected] and
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: CARD, caspase recruitment domain; HA, hemagglutinin; HAV, hepatitis A virus; HBeAg, hepatitis B e-Ag; HBsAg, hepatitis B s-Ag; HBV,
hepatitis B virus; HBX, hepatitis B virus X; HCC, hepatocellular carcinoma; HCV,
hepatitis C virus; IB, immunoblotting; IKK, IkB kinase; IRF, IFN regulatory factor;
MAVS, mitochondrial antiviral signaling; N–RIG-I, RIG-I N-terminal CARD-like domain mutant; poly(dAT:dAT), poly(deoxyadenylate-thymidylate); TM, transmembrane;
VSV, vesicular stomatitis virus; WT, wild-type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903874
(2, 3) and cytosolic sensory molecules, such as the multidomaincontaining NOD proteins, RIG-I, and MDA5 helicases (4–6). Both
RIG-I and MDA5 contain caspase recruitment domains (CARDs)
that interact with the CARD domain-containing protein mitochondrial antiviral signaling (MAVS) upon binding to uncapped RNA,
resulting in MAVS association with IkB kinase (IKK) proteins.
MAVS association with IKKa/b activates NF-kB; its association
with TBK1 as well as IKKε leads to activation of IFN regulatory
factor (IRF)-3. Coordinated activation of NF-kB and IRF-3 pathways leads to the assembly of a multiprotein enhancer complex that
drives expression of IFN-b–and IFN-mediated antiviral immunity
(7–11). As a countermeasure, many viruses have evolved strategies
to interfere with these innate signaling events and thus inhibit
IFN-b production. For example, hepatits C virus (HCV) NS3/4A
protease blocks the RIG-I–mediated signaling pathway by cleaving
the MAVS protein to block IFN-b gene expression (12–17). In
a remarkable parallel to the HCV NS3/4A protease, a stable intermediate product of hepatitis A virus (HAV) polyprotein processing,
3ABC, also targets MAVS for proteolysis (18).
For HBV, however, little is known about the role of the innate
immune system in HBV infection. Wu et al. (19) demonstrated that
activation of the local innate immune system of the liver through
the TLR system was able to effectively suppress HBV replication
in vivo and in vitro. They also showed that hepatitis B s-Ag (HBsAg),
hepatitis B e-Ag (HBeAg), or HBV virions almost completely
abrogated TLR-induced antiviral activity, which correlated with suppression of IFN-b production and subsequent induction of IFNstimulated genes as well as suppressed activation of IRF-3, NF-kB,
and ERK1/2 (20). These studies indicate that HBV can target the
TLR system and thereby attenuate the antiviral response of the innate
immune system. However, the role of the intracellular RIG-I–MAD5
innate immune system in HBV infection remains elusive. Recent
studies have shown that primary hepatocytes retain robust IFN-b
responses to intracellular polyinosinic:polycytidylic acid as well
as to Sendai virus infection (21). Additionally, activation of IFN
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Previous studies have shown that both hepatitis A virus and hepatitis C virus inhibit innate immunity by cleaving the mitochondrial
antiviral signaling (MAVS) protein, an essential component of the virus-activated signaling pathway that activates NF-kB and IFN
regulatory factor-3 to induce the production of type I IFN. For human hepatitis B virus (HBV), hepatitis B s-Ag, hepatitis B e-Ag,
or HBV virions have been shown to suppress TLR-induced antiviral activity with reduced IFN-b production and subsequent
induction of IFN-stimulated genes. However, HBV-mediated suppression of the RIG-I–MDA5 pathway is unknown. In this study,
we found that HBV suppressed poly(deoxyadenylate-thymidylate)-activated IFN-b production in hepatocytes. Specifically,
hepatitis B virus X (HBX) interacted with MAVS and promoted the degradation of MAVS through Lys136 ubiquitin in MAVS
protein, thus preventing the induction of IFN-b. Further analysis of clinical samples revealed that MAVS protein was
downregulated in hepatocellular carcinomas of HBV origin, which correlated with increased sensitivities of primary murine
hepatocytes isolated from HBX knock-in transgenic mice upon vesicular stomatitis virus infections. By establishing a link between
MAVS and HBX, this study suggests that HBV can target the RIG-I signaling by HBX-mediated MAVS downregulation, thereby
attenuating the antiviral response of the innate immune system. The Journal of Immunology, 2010, 185: 1158–1168.
The Journal of Immunology
expression by cytosolic dsDNA also requires the intracellular dsRNA
sensor RIG-I and its adaptor molecule MAVS (22, 23), raising the
possibility that HBV might have also evolved strategies to interrupt the intracellular RIG-I–MAVS signaling pathway.
Among the proteins encoded by HBV, the hepatitis B virus X
(HBX) protein is essential for viral replication in vivo (24) and is
thought to contribute to hepatocarcinogenesis (25–27). HBX
exerts most of its activities through direct interaction with TATAbinding proteins, leucine-zipper proteins, and DNA repair proteins
(28–32). In this study, we report that MAVS interacts with HBX
and is another new target of HBX protein. We found that HBX
promoted the degradation of MAVS, thus preventing the induction
of IFN-b. Further analysis of clinical samples revealed that MAVS
protein was downregulated in hepatocellular carcinomas (HCCs)
of HBV origin, which correlated with increased sensitivities of
primary murine hepatocytes isolated from HBX knock-in transgenic mice upon vesicular stomatitis virus (VSV) infections.
Materials and Methods
HEK293, HepG2, HepG2215, and HepG2-1117 cells were grown in DMEM
(Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS
(HyClone Laboratories, Logan, UT). Cells were treated with MG132
(Sigma-Aldrich, St. Louis, MO) as noted in the text. HepG2-1117 cells
contain 1.05-fold HBV genome (subtype ayw) under inducible Tet-off control (33). Myc-tagged HBX and their mutants, Myc-tagged HBV X, S, S1,
and C, were expressed by cloning the genes into the pcDNA3-based vector
(Invitrogen). HBV DNA genome head-to-tail dimer was provided by Prof.
Y. Wang in our laboratory. HBX-deleted HBV-2 DNA was generated by
two-step PCR using a QuikChange kit (Strategene, La Jolla, CA). GST
fusion proteins were generated by expression in pGEX4T-2-based vectors
(Amersham Biosciences, Piscataway, NJ) in Escherichia coli BL21 (DE3).
Flag-MAVS plasmid was provided by Zhijian Chen (University of Texas
Southwestern Medical Center, Dallas, TX), and NF-kB–Luc, IFN-b–Luc,
or IRF-3–Luc reporter gene constructs were provided by Li Li
(Biotechnology Institute of China, Beijing, China). MAVS mutants were
cloned into pcDNA3 using overlap extension PCR.
Immunoprecipitation and immunoblot analysis
Immunoprecipitation and immunoblot analysis were performed as previously described (34). Anti-MAVS (Abcam, Cambridge, MA), anti-Myc,
HRP-conjugated anti-Flag (Sigma-Aldrich), anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA), or anti–b-tubulin (Sigma-Aldrich) Abs were used.
Subcellular fractionation
Cells were washed 36 h after transfection in hypotonic buffer (10 mM TrisHCl [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, protease inhibitors) and then
homogenized in the same buffer by bouncing 20 times. The homogenate was
centrifuged at 500 3 g for 5 min to remove nuclei and unbroken cells. The
supernatant was centrifuged again at 5000 3 g for 10 min to generate membrane pellets containing mostly mitochondria and cytosolic supernatant.
Protein-binding assays
In GST pull-down experiments, cell lysates were incubated for 2 h at 4˚C
with 5 mg purified GST or GST fusion proteins bound to glutathione beads.
The absorbates were washed with lysis buffer and then subjected to SDSPAGE and immunoblot analysis. An aliquot of the total lysates (5%, v/v)
was included as a loading control on the SDS-PAGE.
Luciferase reporter assays
HepG2 cells were transfected with 0.2 mg of the luciferase reporter pNF-kB–
Luc, IFN-b–Luc, or IRF-3–Luc plus 0.02 mg of the Renilla reporter pRL-TK,
with or without various amounts of MAVS, RIG-I N-terminal CARD-like
domain mutant (N–RIG-I) expression vector, or poly(deoxyadenylatethymidylate) [poly(dAT:dAT)]. Transfected cells were collected and luciferase
activity was assessed as previous described (34). All experiments were repeated at least three times.
RNA analysis
First-strand cDNA was generated from total RNA using random priming
and Moloney murine leukemia virus reverse transcriptase (Invitrogen).
Real-time PCR was performed using QuantiTect SYBR Green PCR Master
Mix (Qiagen, Valencia, CA) in triplicate experiments and analyzed on an ABI
Prism 7700 analyzer (Applied Biosystems, Foster City, CA). All real-time
values were normalized to 18S rRNAwith IFN-b using the following primers:
IFN-b sense, 59-CACGACAGCTCTTTCCATGA-39; IFN-b antisense, 59AGCCAGTGCTCGATGAATCT-39.
In vivo ubiquitination assays
HEK293 cells were cotransfected with plasmids expressing Flag-MAVS,
Myc-HBX, and hemagglutinin (HA)-tagged ubiquitin. Cells were treated
with MG132 (20 mM, 6 h) at 48 h after transfection, and they were then
immunoprecipitated with anti-Flag Ab.
Clinical samples
Liver tumor samples and adjacent noncancerous tissues were obtained from
Shaanxi Chao Ying Biotechnology (Xi’an, Shannxi, China). Immunohistochemistry was performed as previously described (35). Rabbit anti-MAVS
(Abcam) were used as primary Abs.
Mice and cells
HBX knock-in mice were generated as previously described (36). Primary
murine hepatocytes from wild-type (WT) and HBX transgenic mice were
isolated and cultured as described previously (19).
Viral infections
VSV, originally obtained from Dr. W. Chen (Institute of Pathology, Beijing,
China), was harvested from cell culture supernatants of BHK-21 cells, and
virus titers were determined by plaque formation on Vero cells. Primary murine hepatocytes were infected with the equivalent of 10 ml of VSV stock in
serum-free medium (1 ml/well) for 1 h at 37˚C. The infecting medium was
then removed and replaced with 1 ml of normal growth medium. Cell supernatants were recovered 24 h postinfection, and virus titers were determined
by plaque formation on Vero cells. HepG2-1117 cells were infected for 20 h
with VSV (multiplicity of infection of 0.002) but were not killed; cells were
fixed and then stained with amino black.
Results
Activation of IFN-b expression by cytosolic dsDNA is blocked
by HBV
Recent reportssuggest thatHAVor HCVinfection blocks the induction
of IFN-b synthesis by dislodging MAVS from the mitochondria. To
determine whether RIG-I–MDA5 signaling was disrupted by HBV
infection, we ectopically expressed N–RIG-I or MAVS together with
a luciferase reporter driven by the IFN-b promoter (IFN-b–Luc) in
HepG2 cells transfected with a head-to-tail dimer of HBV genome
(HBV-2 DNA). Previous studies have reported that HBV-2 DNA
transfection supports the production of HBV particles in HepG2 cells.
When using HepG2 cells, we also found that cells transfected with
HBV-2 DNA continuously secreted HBsAg into the supernatant
(Supplemental Fig. 1), suggesting that the transfected HBV DNA
could replicate and produce virus particles. We thus analyzed whether
HBV could affect type I IFN signaling using the HBV DNAtransfected HepG2 cells. As shown in Fig. 1A, both MAVS and
N–RIG-I activated the IFN-b promoter in HepG2 cells transfected
with empty vector, whereas these responses were substantially
reduced in HepG2 cells transfected with the HBV-2 DNA. These
results suggest that HBV DNA blocks RIG-I–MDA5 signaling.
Poly(dAT:dAT) is a synthetic dsDNA that mimics dsDNA virus,
which has previously been shown to induce IFN-b through RIG-I–
MDA5 (11). In agreement with this observation, we also demonstrated that IFN-b response to the transfected poly(dAT:dAT) or
VSV infection was reduced by HBV DNA (Fig. 1A). Furthermore,
we observed that HBV DNA suppressed the activation of NF-kB and
IRF-3 reporters through MAVS, RIG-I, poly(dAT:dAT), and VSV
infection (Fig. 1A). To better understand the interference of IFN-b
signaling by HBV, 105 HepG2 monolayer cells in a 6-cm plate were
infected with HBV-positive serum containing 107 copies/ml HBVs or
with control serum from uninfected individuals. Cells were washed
eight times to remove the excess of viral inputs 2 h postinfection
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Cell culture and transfections
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HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY
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FIGURE 1. HBV DNA blocks IFN-b induction by MAVS. A, HepG2 cells were transfected with plasmid-expressing HBV-2 DNA. After 36 h, cells were
transfected with N–RIG-I, MAVS, or poly(dAT:dAT) together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. Cells transfected with HBV-2 DNA together with
IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc were infected with VSV for 20 h. The Luc activity was measured 24 h later and normalized for transfection efficiency.
B, HepG2 cells were infected with HBV-positive serum containing 107 copies/ml HBV. Cells were washed eight times to remove excess viral inputs 2 h
postinfection and were then transfected with poly(dAT:dAT) together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. Serum from uninfected individuals was
used as a control. The Luc activity was measured 24 h later and normalized for transfection efficiency. C, HepG2-1117 cells were infected with VSV with or
without doxcycline. Protein extracts were then resolved by SDS-PAGE (upper panel) or native gel electrophoresis (lower panel). Phosphorylation or dimerization of IRF-3 was detected by IB with an IRF-3 Ab. D, The experiments were carried out as in B, except that HBV X, S, S1, and C gene expression
plasmid was transfected together with poly(dAT:dAT). Cell lysates were immunoblotted with anti-Flag Ab. The Luc activity was measured 24 h later and
normalized for transfection efficiency. E, HepG2 cells were transfected with HBV–2-DNA or HBV-2 DNA-4HBX. After 36 h, cells were transfected with poly
(dAT:dAT) together with IFN-b–Luc. The Luc activity was measured 24 h later and normalized for transfection efficiency. Cell lysates were immunoblotted
with anti-HBX Ab. F, HepG2 cells were transected with plasmids expressing HBX together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc and were then were
infected with VSV. The Luc activity was measured 24 h later and normalized for transfection efficiency. G, HepG2 cells were transfected with the HBV-2 DNA
or HBX expression plasmid together with the expression vectors for RIG-I, DAI, MAVS, TBK1, or IKKε. Cell lysates were immunoblotted with anti-Flag or
anti-Myc Ab. b-Tubulin was used as an equal loading control. For A, B, and D–F, the Luc activity was measured 24 h later and normalized for transfection
efficiency. The error bar represents SD from the mean value of duplicated experiments. IB, immunoblotting.
The Journal of Immunology
and were then transfected with poly(dAT:dAT) together with IFN-b–
luc, NF-kB–Luc, or IRF-3–Luc. We found that HBV also inhibited
the induction of IFN-b, IRF-3, and NF-kB by cytosolic poly(dAT:
dAT) (Fig. 1B). HepG2-1117 cells contain Tet-off HBV-1.05 genome. Supplemental Fig. 1C shows that IFN-b, IRF-3, and NF-kB
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levels induced by VSV were lower in HepG2-1117 cells without
doxcycline. In agreement with these data, immunoblotting experiments also showed that VSV-activated IRF-3 phosphorylation was
inhibited by HBVand in HepG2-1117 cells without doxcycline (Supplemental Fig. 1B, 1C). To delineate the mechanism underlying the
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FIGURE 2. HBX associates with MAVS. A, HEK293 cells were transfected with Flag-MAVS–expressing plasmid. The GST-fusion protein absorbates from
cell lysates were analyzed by immunoblotting with anti-Flag Ab (top panel). Loading of the GST proteins was assessed by Coomassie blue staining (bottom
panel). B, HEK293 cells were cotransfected with Flag-MAVS and Myc-HBX expression plasmid or Flag-vector, and anti-Flag or IgG immunoprecipitates were
analyzed by immunoblotting with anti-Myc or anti-Flag Ab. C, HEK293 cells were transfected with Flag-HBX expression plasmid or Flag-vector, and anti-Flag
immunoprecipitates were analyzed by immunoblotting with anti-MAVS or anti-Flag Ab. Lysates from HEK293 cells transfected with HBV-2 DNAwere subjected
to immunoprecipitation with anti-MAVS or IgG, fractionated by SDS-PAGE, and subsequently analyzed by immunoblotting with anti-MAVS Ab or anti-HBX Ab.
D, HEK293 cells were cotransfected with Myc-HBX expression plasmid and Flag-MAVS or with Flag-MAVS mutants, and anti-Flag immunoprecipitates were
analyzed by immunoblotting with anti-Myc or anti-Flag Ab. E, HEK293 cells were cotransfected with Flag-MAVS expression plasmid and Myc-HBX or with
Myc-HBX mutants, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab. F, HEK293 cells were transfected with
Myc-HBX (73–154) or Myc-HBX (73–154 C115A) plasmid, and mitochondria and nuclear and cytosolic fractions were analyzed by immunoblotting with antiMyc, cytochrome c, or lamin B1 Ab. G, HEK293 cells were cotransfected with Flag-MAVS expression plasmid and Myc-HBX (73–154) or with Myc-HBX (73–
154 C115A) plasmid, and anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag Ab.
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reduced IFN-b signaling, expression constructs for four HBVencoded proteins (X, S, S1, and C) were generated, and one of these
four constructs was used to cotransfect HepG2 cells together with
poly(dAT:dAT). As shown in Fig. 1D, the induction of IFN-b by
poly(dAT:dAT) was only inhibited by HBX. To confirm the
inhibitory role of HBX in IFN-b signaling, we deleted the HBX
gene in HBV-2 DNA. We found substantially higher levels of IFN-b
induction in the cells thus transfected compared with those
transfected with HBV-2 DNA (Fig. 1E). HBX introduction also
blocked IFN-b promoter activation by VSV induction (Fig. 1F),
suggesting that HBX was able to independently inhibit IFN-b
activation.
To determine the modulation of protein abundance of the RIG-I–
MAVS pathway by HBV DNA and HBX, immunoblotting was
used to analyze the levels of RIG-I, MAVS, DAI, IKKε, and
HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY
TBK1 proteins from HEK293 cells transfected with either empty
vector, HBV-2 DNA, or HBX expression plasmid. As shown in
Fig. 1G, MAVS protein abundance was substantially reduced by
HBV-2 DNA or HBX, suggesting that MAVS is a target of HBV
and that HBX is a potential factor that may interfere with the
stability of MAVS and thus IFN-b signaling.
HBX interacts with MAVS
Our observation that HBX could downregulate MAVS, as well as
the fact that both MAVS and some portion of HBX reside in the
mitochondria compartment, raised the possibility that HBX might
physically interact with MAVS. To test this possibility, lysates from
HEK293 cells were incubated with GSTor GST-HBX fusion protein.
We found that MAVS bound to GST-HBX but not to GST (Fig. 2A),
demonstrating an in vitro interaction between HBX and MAVS.
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FIGURE 3. HBX blocks IFN-b induction by MAVS. A, HEK293 cells were transfected with increasing amounts of HBX expression vector. After 36 h,
cells were transfected with MAVS together with IFN-b–Luc, NF-kB–Luc, or IRF-3–Luc. The Luc activity was measured 24 h later and normalized for
transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. B, The experiments were carried out as in A, except that
N–RIG-I plasmid was transfected in lieu of MAVS. C, HEK293 cells were transfected with plasmid-expressing HBX. After 36 h, cells were transfected
with poly(dAT:dAT). RNA was extracted and IFN-b mRNA was analyzed by quantitative RT-PCR. D, The experiments were carried out as in A, except that
IKKε plasmid was transfected in lieu of MAVS.
The Journal of Immunology
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FIGURE 4. HBX downregulates MAVS. A, HEK293 cells were transfected with plasmids expressing increasing amount of Myc-HBX (0.25, 0.5, and
0.75 mg). Whole-cell lysates were analyzed by immunoblotting with anti-MAVS or anti-Myc Ab. b-Tubulin was used as an equal loading control. B, Myc-HBX
or Myc-HBX 73–154 C115A plasmid was transfected with the expression vector encoding HA-tagged ubiquitin. Cells were grown in DMEM containing
MG132 (20 mM) for 6 h. Anti-MAVS immunoprecipitates were analyzed by immunoblotting with anti-HA Ab, whole-cell lysates were subjected to
immunoblotting with anti-Myc and anti-MAVS Ab, and b-tubulin was used as an equal loading control. C, Myc-HBX or empty vectors were cotransfected
with plasmids encoding Flag-MAVS or its mutants. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-Myc Ab. Ten nanograms of
plasmid encoding GFP was transfected as a loading control, and whole-cell lysates were analyzed by immunoblotting with anti-GFP Ab. D, Myc-HBX or
empty vectors were cotransfected with plasmids encoding Flag-MAVS or its mutants together with IFN-b–Luc. The Luc activity was measured 24 h later and
normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments. E, Myc-HBX was transfected with the
expression vector encoding Flag-MAVS or Flag-MAVS K136R mutant together with HA-tagged ubiquitin. Cells were grown in DMEM containing MG132
(20 mM) for 6 h. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-HA Ab, whole-cell lysates were subjected to immunoblotting with
anti-Myc and anti-Flag Ab, and b-tubulin was used as an equal loading control. F, HEK293 cells were cotransfected with plasmids expressing Flag-MAVS
and Myc-HBX or its mutants. Whole-cell lysates were analyzed by immunoblotting with anti-Flag or anti-Myc Ab, and b-tubulin was used as an equal loading
control. G, HEK293 cells were cotransfected with Flag-MAVS, Myc-HBX, or HBX mutants together with IFN-b–Luc. The Luc activity was measured 24 h
later and normalized for transfection efficiency. The error bar represents SD from the mean value of duplicated experiments.
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HBX blocks MAVS-mediated IFN-b induction
To better understand how HBX blocks IFN-b signaling through
MAVS, increasing amounts of HBX expression vector and 0.5 mg
of MAVS expression construct were transfected into HEK293 cells
together with IFN-b–Luc. As was shown in Fig. 3A, as low as 0.5 mg
of HBX expression construct was sufficient to exert a potent
repression of IFN-b response, and the extent of repression
increased with increasing amounts of HBX expression, suggesting
that HBX inhibited the induction of IFN-b by MAVS in a dosedependent manner. We also observed similar repression of MAVSinduced activation of NF-kB and IRF-3 reporters by HBX (Fig. 3A).
We then transfected cells with poly(dAT:dAT) or with an expression
vector encoding N–RIG-I together with HBX. Fig. 3B shows that the
induction of IFN-b by RIG-I or poly(dAT:dAT) (data not shown)
was also inhibited by HBX. As expected, IFN-b mRNA levels were
reduced sharply in cells containing the HBX expression plasmid
(Fig. 3C). In contrast, the induction of IFN-b by IKKε was not
affected (Fig. 3D). Collectively, these data suggest that HBX
inhibits RIG-I–MAVS signaling at the level of MAVS or at a step
downstream of MAVS, but upstream of IKKε.
HBX downregulates MAVS
To further elucidate the mechanism for the inhibitory effect of
HBX on antiviral signaling, we examined the effect of HBX on the
endogenous MAVS protein level. Increasing amounts of the HBX
expression vector were transfected into HEK293 cells. A striking
reduction in the abundance of endogenous MAVS with overexpressed HBX was found, and this reduction of MAVS by HBX was
also dose-dependent (Fig. 4A). As a control, increasing amounts of
HBV S Ag expression vector did not change the level of endogenous MAVS (data not shown), indicating that this effect is specific to HBX. Similarly, the HepG2-1117 cell line, which contains
an inducible Tet-off HBV-1.05 genome, showed reduced endogenous MAVS levels without doxcyline. Quantitative RT-PCR
revealed that HBX overexpression did not change MAVS mRNA
levels (data not shown), suggesting that HBX downregulates MAVS
by posttranscriptional modification. Indeed, the estimated half-life
of the MAVS was significantly longer than that of MAVS in the
presence of HBX (data not shown). To further delineate the mechanisms responsible for the HBX-mediated MAVS degradation,
HEK293 cells were cotransfected with plasmids expressing MycHBX or Myc-HBX 73–154 C115A together with HA-ubiquitin in
the presence of proteasome inhibitors MG132 (20 mM for 6 h), and
MAVS was then immunoprecipitated by anti-MAVS Ab and blotted
with anti-HA Ab. As shown in Fig. 4B, WT HBX introduction led
to an increased steady level of MAVS ubiquitination whereas HBX
73–154 C115A abolished this effect. These results indicate that
HBX may have a major role in MAVS ubiquition and its proteasome-mediated degradation.
To further dissect the possible HBX-mediated ubiquitination sites
in MAVS, 14 mutants were generated by substituting the 14 lysine residues on the MAVS molecule individually with arginine (KRR) and
then tested for their stability by introducing them with HBX into
HEK293 cells. As shown in Fig. 4C, HBX had little effect on the
cellular abundance of MAVS K136R mutations and modestly decreased the cellular abundance of MAVS K297R and K420R mutation. In contrast, HBX led to a sharp decrease on other MAVS KRR
mutations and WT MAVS as well. Furthermore, MAVS K136Rinduced IFN-b activation was only partially inhibited by HBX. The
extent of HBX suppression on MAVS K297R and K420R mutationinduced IFN-b activation was much lower than that achieved with
WT MAVS or other MAVS KRR mutations (Fig. 4D). In agreement
with this observation, HBX induced increased ubiquitin levels of
WT MAVS with less effect on the MAVS K136R mutant (Fig. 4E).
These results suggest that Lys136 is one of the critical sites for
HBX-mediated ubiquitination in MAVS proteins.
FIGURE 5. HBX affects the kinetics of RIG-I–MAVS signaling. HEK293
cells were cotransfected with Myc-MAVS and Flag–RIG-I expression plasmids together with the expression vector encoding GFP-HBX or empty
vector. After 24 h, cells were treated with VSV for indicated times. Cells
were then harvested at different times after VSV infection as indicated. AntiFlag or IgG immunoprecipitates were analyzed by immunoblotting with antiMyc or anti-Flag Ab. HBX expression was monitored by immunoblotting
using GFP Ab. b-Tubulin was used as an equal loading control.
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To test if HBX binds to MAVS in mammalian cells, HEK293 cells
were transfected with Myc-tagged HBX and Flag-tagged MAVS.
Immunoblotting analysis of anti-Flag immunoprecipitates with antiMyc Ab showed a significant association between Flag-MAVS and
Myc-HBX (Fig. 2B). To further characterize the endogenous MAVS
interaction with HBX, lysates from HEK293 cells transfected with
Flag-HBX were subjected to immunoprecipitation with anti-Flag
Ab by immunoblotting with an anti-MAVS Ab, and MAVS was
found in Flag-HBX–transfected immunoprecipitates but not in the
nontransfected control immunoprecipitations (Fig. 2C). Additionally, lysates from HEK293 cells transfected with HBV-2 DNA were
subjected to immunoprecipitation. HBX was shown to interact with
endogenous MAVS in the HBV-2 DNA-transfected cells (Fig. 2C),
suggesting that HBX interacts with MAVS in vivo.
To map the domains of MAVS responsible for the interaction with
HBX, Flag-tagged proteins containing a deletion of various regions
of MAVS (CARD, pro, and transmembrane [TM] domain) were
prepared, and the ability of each of these mutants to interact with
HBX was analyzed by immunoprecipitation. Fig. 2D shows that
Myc-tagged HBX interacted with full-length and pro mutant MAVS
but not with the TM mutant MAVS. The CARD MAVS mutant also
interacted with HBX with lower affinity. Thus, the interaction with
HBX is specific for the CARD and TM domains of MAVS.
To define the interacting regions of HBX on MAVS, Myc-tagged
proteins containing N-terminal and C-terminal HBX were prepared,
and the ability of each of these proteins to interact with MAVS was
analyzed by immunoprecipitation. The C-terminal 73–154 residues
of HBX did not affect its ability to interact with MAVS, whereas
the N-terminal HBX 1–72 domain totally eliminated its ability to
bind MAVS (Fig. 2E). Thus, the interaction with MAVS requires
the C-terminal domain of HBX. A previous report revealed the key
amino acid for mitochondrial targeting was mapped to be HBX C
terminus 111–116 aas (37); when Cys115 of HBX was mutated to
alanine (HBX C115A), the mitochondrial targeting property of
HBX was abrogated. Consistent with this report, we found that
when Cys115 of 73–154 C-terminal HBX was substituted with alanine (HBX 73–154 C115A), its mitochondria localization was partially abrogated and its interaction with MAVS was much weaker
than for the intact HBX C-terminal (HBX 73–154) (Fig. 2F, 2G).
HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY
The Journal of Immunology
We also examined whether HBX 73–154 C115A could also
affect the protein stability of MAVS. In contrast to the HBX
73–154 C-terminal domain, the cellular abundance of MAVS in
73–154 C115A-expressing cells was unchanged (Fig. 4F). Additionally, the extent of MAVS-induced IFN-b repression by HBX
73–154 C115A was lower than that achieved with WT HBX or
with the HBX 73–154 C-terminal domain (Fig. 4G). These results
indicate that interaction between HBX and MAVS is responsible
1165
for MAVS downregulation and MAVS-mediated antiviral signaling
inhibition.
HBX affects the kinetics of RIG-I–MAVS signaling
Our studies have shown that HBX inhibits RIG-I–MAVS signaling
by downregulating MAVS, indicating that HBX might affect the
kinetics of the MAVS–RIG-I association upon virus infection.
HEK293 cells were cotransfected with Myc-MAVS and Flag–RIG-I
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FIGURE 6. Expressions of MAVS proteins in liver cancer patients and their association with HBV disease. A, HepG2, HepG2215, and primary murine
hepatocytes isolated from HBX transgenic mice or control mice were cultured, and whole-cell lysates were analyzed by immunoblotting with anti-MAVS.
b-Tubulin was used as an equal loading control. HepG2-1117 cells containing 1.05-fold HBV genome under Tet-off control were cultured with or without
doxcycline for 7 d, whole-cell lysates were analyzed by immunoblotting with anti-MAVS, and b-tubulin was used as an equal loading control. HepG2-1117 cells
were cultured with or without doxcycline for 7 d and transfected with Flag-MAVS together with HA-Ub plasmid, anti-Flag immunoprecipitates were analyzed by
immunoblotting with anti-HA Ab, whole-cell lysates were subjected to immunoblotting with anti-Flag Ab, and b-tubulin was used as an equal loading control. B,
Representative immunoblot of MAVS protein in HBV-induced HCC (C1–C4), HBV-irrelevant HCC (C5–C8), and in their matched controls (N1–N4). Immunoblot of HBX expression was also analyzed in C1–C8 samples. C, Representative immunohistochemical staining of MAVS expression in normal donor livers (1),
HBV-irrelevant cirrhosis (2), HBV-irrelevant HCC (3), HBV-induced cirrhosis (4), and in HBV-induced HCC (5). 1–5, Original magnification 3200.
1166
expression plasmids together with the expression vector encoding
GFP-HBX or empty vector. After 24 h, cells were treated with VSV
and then harvested at different times as indicated. As shown in
Fig. 5, the interaction of MAVS and RIG-I was enhanced by VSV
infection, whereas the introduction of HBX not only eliminated
this enhancement but it also reduced MAVS–RIG-I interaction. Because this RIG-I–MAVS interaction is required for RIG-I–mediated
IFN-b signaling, the disruption of this interaction by HBX would
squelch downstream signaling. Collectively, these observations
solidify the negative regulatory role of HBX in MAVS-mediated
antiviral responses, and they demonstrate that HBX exerts its inhibition of virus induced RIG-I–MAVS signaling through downregulation of MAVS.
Expression of MAVS proteins in liver cancer patients and their
association with HBV disease
HCC, 86% expressed lower levels of MAVS (13/15), suggesting
a negative correlation between HBX expression and MAVS protein level.
MAVS protein levels were also measured by immunohistochemistry in normal donor livers (n = 14), in livers of HBVunrelated cirrhosis and HCC (n = 30), in livers with cirrhosis
caused by HBV infection (n = 22), and in HCC caused by HBV
infections (n = 24). Results showed that 78.6% (11/14) of healthy
livers, 76.6% (23/30) of HBV-unrelated liver diseases, and 30.4%
(14/46) of HBV-related liver diseases stained strongly positive for
MAVS proteins (Fig. 6C). The overall differences in MAVSpositive staining between HBV-induced liver disease groups and
the other three HBV-irrelevant groups including normal controls
were significant (30.4 versus 77.6%). Taken together, these data
suggest a negative correlation between MAVS protein and HBV
liver disease.
HBX regulates RIG-I-dependent antiviral cellular responses
We next sought to determine whether HBX regulates replication of
VSV virus replication, as RIG-I–mediated IFN-b signaling is
critical in restricting replication of these RNA viruses. We
therefore used VSV to infect HepG2-1117 cells with or without
doxcycline. The results showed that HepG2-1117 without doxcycline increased the production of VSV ~50% (Fig. 7A). Furthermore, primary murine hepatocytes isolated from HBX knock-in
mice or the WT littermate control mice were cultured and infected
with VSV virus. Fig. 7B shows that exogenous expression of HBX
increased the production of infectious VSV. Taken together, these
results provide more evidence that HBX modulates the innate
antiviral cellular response by acting as a negative regulator of
RIG-I–mediated IFN-b signaling infection.
Discussion
The targeting of MAVS by both the cysteine protease of HAV and
the serine protease of HCV represents a remarkable example of
convergent virus evolution (38) and provides strong evidence for
the importance of MAVS to host control of virus infections in the
liver. In this study, we show that both HBV DNA or HBV suppressed
poly(dAT:dAT)-activated IFN-b production in hepatocytes. Additionally, MAVS interacts with HBX and is indeed the target of HBX
protein. Specifically, we found that HBX promoted the degradation
FIGURE 7. HBX regulates RIG-I–dependent antiviral cellular responses. A, HepG2-1117 cells with or without doxcycline were infected for 20 h with
VSV (multiplicity of infection of 0.002) but not killed; cells were fixed and then stained with amino black. B, Primary murine hepatocytes were infected
with the equivalent of 10 ml of VSV stock in serum-free medium (1 ml/well) for 1 h at 37˚C. The infecting medium was then removed and replaced with
1 ml of normal growth medium. Cell supernatants were recovered 24 h postinfection, and virus titers were determined by plaque formation.
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To examine the status of MAVS in the presence of HBV, we
performed Western blotting analysis to examine the expression of
MAVS protein levels in HepG2-derived, HBV DNA-transfected
HepG2215 cells, HepG2-1117 cells that contain Tet-off HBV1.05 genome, and in primary murine hepatocytes isolated from
HBX transgenic mice. It is evident that MAVS was present at
low levels in HepG2215 cells and in HepG2-1117 cells without
doxcycline; additionally, transfected MAVS in HepG2-1117 cells
showed increased ubiquitin modification when HBX expression
was induced without doxcycline (Fig. 6A). Consistent with this
finding, primary hepatocytes isolated form HBX knock-in transgenic mice also showed reduced endogenous MAVS level compared with WT littermate control mice (Fig. 6A).
We next examined the expression of MAVS proteins in HBVinduced HCC (n = 20) and matched healthy controls (n = 20), as
well as in HBV-unrelated HCC (n = 20) and matched healthy
controls (n = 20). Our results showed that 80% (16/20) of HBVinduced HCC expressed lower levels of MAVS (Fig. 6B) and that
10% (2/20) of HBV-unrelated HCC expressed lower levels of
MAVS (Fig. 6B). The occurrence of reduced MAVS expression
between the HBV-induced HCC and the HBV-unrelated HCC was
significant. To further delineate the correlation between HBX and
MAVS, HBX protein expression was also analyzed in those 20
HBV-induced HCC samples. Seventy-five percent (15/20) showed
positive HBX expression (Fig. 6B); among the 15 HBX-positive
HBX DESTABILIZES MAVS TO EVADE INNATE IMMUNITY
The Journal of Immunology
characterized by the production of type I IFN-a/b cytokines and the
activation of NK cells. HBV replication can be efficiently limited
by a and b IFN, but data on acutely infected chimpanzees suggest
that such antiviral cytokines are not triggered by HBV replication
(44). A further characteristic of HBV in relationship to early host
defense mechanisms resides in the lack of IFN-a and IFN-b production. HBV might have evolved strategies to escape the initial
antiviral defense mechanisms activated by the TLR system or RIGI–MDA5 pathway. Previous reports have shown that HBV almost
completely abrogated TLR-induced antiviral activity (20). Cheng
et al. (45) demonstrated that recombinant HBsAg inhibits LPS/
TLR4-induced NF-kB activation, leading to reduced COX-2, IL18, and IFN-g production in the human monocytic cell line THP-1.
Other studies have shown lower TNF-a levels in HBeAg-positive
patients compared with HBeAg-negative patients (46, 47). In this
study, we analyzed the involvement of MAVS in HBV infection by
using liver samples from HBV-induced cirrhosis and from HCC
patients. We demonstrated that MAVS was downregulated markedly in livers from these patients, with the decrease in MAVS
having been correlated with HBX expression in HCC liver tumors.
Several studies have demonstrated the pivotal position of RIG-I–
MAVS signaling in host responses against a number of viruses (48,
49). Recent research has indicated that the induction of TLR- and
RIG-I–MDA5-mediated host cellular innate immune responses by
overexpression of the three pattern recognition receptors adaptors,
IPS-1, TRIF, and MyD88, dramatically reduces the levels of HBV
mRNA and DNA in both HepG2 and Huh7 cells. These observations provide more evidence that HBV might evolve multiple stagiest to evade TLR-dependent and -independent signaling pathways;
further understanding of the nature of these mechanisms should
yield novel strategies for developing antivirals that evoke responses
to eliminate HBV infection.
Acknowledgments
We thank Zhijian Chen (University of Texas Southwestern Medical Center,
Dallas, TX) for providing the MAVS plasmid.
Disclosures
The authors have no financial conflicts of interest.
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of MAVS through Lys136 ubiquitin in MAVS protein, thus preventing the induction of IFN-b. We also show that the enhanced
interaction of MAVS and RIG-I by VSV infection was eliminated
in the presence of HBX. Meanwhile, MAVS protein abundance was
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response of the innate immune system.
HBX, a virally encoded protein of 154 aas, has been shown to be
essential for the establishment of HBV infection in vivo. Its gene
product also activates a variety of viral and cellular promoters in
diverse cell types. Although HBX does not bind to dsDNA, it does
regulate transcription of a variety of cellular and viral genes by
interacting with cellular proteins and/or components of signal
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the antiviral pathway through different mechanisms under different stimuli.
MAVS contains an N-terminal CARD-like domain and
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outer mitochondrial membrane. The mitochondrial-targeting transmembrane domain is essential for MAVS signaling, thus implicating a new role for mitochondria in innate immunity. Previous
reports have shown that HBX regulates fundamental aspects of
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a mitochondrial protein that is essential for innate immunity, thus
shedding new light on the physiological significance of HBX, which
may contribute to liver disease associated with HBV-persistent
infection. Previous reports have also shown that MAVS proteins
undergo proteasomal degradation by ubiquitin E3 ligase RNF125mediated ubiquitin conjugation (41). In this study, we demonstrate
that HBX promotes MAVS ubiquitin to trigger its proteasomemediated degradation through the Lys136 site, and that MAVS
K136R elicits a higher level of IFN-b activation compared with
WT MAVS, suggesting that the MAVS Lys136 site could be the
ubiquitination site of RNF125.
The proteasome is involved in both ubiquitin-dependent and
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on cellular regulatory proteins and viral proteins that interact with
subunits of the proteasome complex and participate in proteasomedependent regulation (42, 43). PSMA7 is a subunit of proteasome
that regulates the activity of this complex associated with HBX,
suggesting that HBX may modulate the function of proteasome by
interacting with PSMA7. Our previous results have shown that
PSMA7 may regulate host innate immune signaling by destabilizing MAVS, raising the possibility that HBX may be potentially
bridged by PSMA7 on the mitochondrial outer membrane to exert
its inhibitory effect on innate immune response, rather than exert
a direct effect.
Although the liver is a particularly important site of persistent
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immune signaling pathways function in hepatocytes. Regardless,
there is strong, albeit indirect, evidence that type I IFN responses
are important in the pathogenesis of chronic viral hepatitis. Innate
host responses during the early phases of viral infections are mainly
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